Exopolysaccharide from marine microalgae belonging to the Glossomastix genus: fragile gel behavior and suspension stability.

With the aim to find new polysaccharides of rheological interest with innovated properties, rhamnofucans produced as exopolysaccharides (EPS) in a photobioreactor (PBR) and an airlift bioreactor (ABR) by the marine microalgae Glossomastix sp. RCC3707 and RCC3688 were fully studied. Chemical characterizations have been conducted (UHPLC - MS HR). Analyses by size-exclusion chromatography (SEC) coupled online with a multiangle light scattering detector (MALS) and a differential refractive index detector showed the presence of large structures with molar masses higher than 106 g.mol-1. The rheological studies of these EPS solutions, conducted at different concentrations and salinities, have evidenced interesting and rare behavior characteristic of weak and fragile hydrogels i.e. gel behavior with very low elastic moduli (between 10-2 and 10 Pa) and yield stresses (between 10-2 and 2 Pa) according to the EPS source, concentration, and salinity. These results were confirmed by diffusing wave spectroscopy. Finally, as one of potential application, solutions of EPS from Glossomastix sp. have evidenced very good properties as anti-settling stabilizers, using microcrystalline cellulose particles as model, studied by multiple light scattering (MLS) with utilization in cosmetic or food industry. Compared to alginate solution with same viscosity for which sedimentation is observed over few hours, microalgae EPS leads to a stable suspension over few days.

Role of some structural features in EPS from microalgae stimulating collagen production by human dermal fibroblasts.

Exopolysaccharides (EPS) from the microalgae Porphyridium cruentum, Chrysotila dentata, Pavlova sp., Diacronema sp., Glossomastix sp., Phaeodactylum tricornutum, and Synechococcus sp. were isolated and depolymerized. First, EPS were submitted to a high pressure pre-treatment step, followed by a solid acid-catalyzed hydrolysis step carried out in a batch or recycle fixed-bed reactor, using a strong acidic cation-exchange resin. Twenty-eight different EPS forms were thus obtained. After characterization of their main structural features (weight- and number-averaged molecular weight, polydispersity index, sulfate and uronic acid contents), we investigated the structure-function relationship of their pro-collagen activity. We found that native microalgae EPS were able to inhibit until 27% of human matrix metalloproteinase-1 (MMP-1) activity while the depolymerized forms were able to enhance collagen production by two different human fibroblast lines, used as cell models due to their major role in dermal collagen biosynthesis. The most active EPS forms, obtained by depolymerization in the recycle fixed-bed reactor of D. ennorea and Glossomastix sp. EPS, led to 390% increase in collagen production. Finally, principal component (PCA) and Pearson analyses indicated that MMP-1 inhibition was strongly correlated to the sulfate group content of EPS whereas collagen production by fibroblasts was mostly related to their proportion of low molecular weight polysaccharides (<10 kDa). Uronic acid content of EPS was also shown essential but only if the size of EPS was reduced in the first place. Altogether, these results gave new insights of the dermo-cosmetic potential of microalgae EPS as well as the key parameters of their activity.

Six new and original EPS from microalgae were isolated.Microalgae EPS were depolymerized by high pressure/solid acid-catalyzed hydrolysis.Native EPS inhibited human matrix metalloproteinase-1.Depolymerized EPS highly stimulated collagen production by human dermal fibroblasts.Structure­function studies revealed that sulfate groups and MW of EPS were crucial.

Exopolysaccharides from Microalgae and Cyanobacteria: Diversity of Strains, Production Strategies, and Applications.

Microalgae and cyanobacteria are photosynthetic organisms that can produce/accumulate biomolecules with industrial interest. Among these molecules, EPSs are macromolecular polysaccharidic compounds that present biological activities and physico-chemical properties, allowing to consider their valorization in diverse commercial markets, such as cosmetic, therapeutic, nutraceutic, or hydrocolloids areas. The number of microalgae and cyanobacteria strains described to produce such EPSs has increased in recent years as, among the 256 producing strains gathered in this review, 86 were published in the last 10 years (~33%). Moreover, with the rise of research on microalgae EPSs, a variety of monosaccharides compositions have been discovered, highlighting the versatility of these organisms. If some production strategies can be applied to increase EPS production yields, it appears that case by case studies are needed to promote EPS synthesis by a strain, as many responses exist. This paper proposes an up-to-date state of the art of the diversity of microalgae and cyanobacteria EPS-producing strains, associated to the variability of compositions. The strategies for the production and extraction of the polymers are also discussed. Finally, an overview of the biological activities and physico-chemical properties allow one to consider their use on several commercial markets.

Exploring the Diversity of Red Microalgae for Exopolysaccharide Production.

Microalgae constitute a remarkable biological diversity but a limited number of them have been the object of study for their ability to produce exoplysaccharides (EPS). Among them, the red marine microalgae Porphyridium or Rhodella produce sulphated EPS, exhibiting some biological activities with potential interest in the pharmaceutical and cosmetic industries. EPS from Porphyridium and Rhodella being relatively similar in their composition, it has long been considered that all the red microalgae produced similar EPS and no attention was paid to other red microalgae. The objective of our work was then to explore the diversity of red microalgae for the production of EPS, focusing in this first step on the screening of the strains for their ability to produce EPS and preliminary structural characterization. The study was conducted with 11 microalgae strains belonging to the proteorhodophytina subphylum. All microalgae were able to produce EPS, released in the culture medium (strains belonging to Porphyridiophyceae and Rhodellophyceae classes) or remaining bound to the cells (strains from Stylonematophyceae class). The analysis of monosaccharides composition was found significantly different, with for instance high levels of glucuronic acids in the EPS from C. japonica and N. cyanea, but also strong differences in the sulphation degrees of polymers (between 1.2 and 28.7% eq. SO4).

A New, Quick, and Simple Protocol to Evaluate Microalgae Polysaccharide Composition.

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides' (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.

Potential of Exopolysaccharide from Porphyridium marinum to Contend with Bacterial Proliferation, Biofilm Formation, and Breast Cancer.

Exopolysaccharide (EPS) from marine microalgae are promising sources of a new generation of drugs. However, lot of them remain to be discovered and tested. In this study, EPS produced by Porphyridium marinum and its oligomers prepared by High Pressure Homogenizer have been tested for different biological activities, i.e., antibacterial, anti-fungal and antibiofilm activities on Candida albicans, as well as for their effects on the viability of murine breast cancer cells. Results have shown that all EPS samples present some biological activity. For antibacterial and antibiofilm activities, the native EPS exhibited a better efficiency with Minimum Inhibitory Concentration (MIC) from 62.5 µg/mL to 1000 µg/mL depending on the bacterial strain. For Candida albicans, the biofilm formation was reduced by about 90% by using only a 31.3 µg/mL concentration. Concerning breast cancer cells, lower molar masses fractions appeared to be more efficient, with a reduction of viability of up to 55%. Finally, analyses of polymers composition and viscosity measurements were conducted on all samples, in order to propose hypotheses involving the activities caused by the intrinsic properties of polymers.

Polyethyleneimine as a tool for compounds fractionation by flocculation in a microalgae biorefinery context.

In the context of emerging biorefinery for microalgae, polyethyleneimine (PEI), has been tested in order to achieve separation of fat-soluble and water-soluble compounds from Haematococcus pluvialis. Several parameters were taken into account (ratio between sample and PEI, pH, and ionic strength) and 2 conditions (0.075% PEI pH 7.4, and 0.100% PEI pH8.5) were studied for up-scalability, with a recovery of flocculated compounds (lipids and pigments), and a complete characterization of both phases. Using 0.075% PEI, pH7.4, 100% sugars and 89.8% proteins were retained in the supernatant, but some trace of beta-carotene were also detected. For 0.100% PEI, pH 8.5, a loss in proteins content was highlighted (61.2% proteins retained), but no residual lipids or pigments were detected. PEI could therefore be considered as an efficient method to fractionate fat-soluble and water-soluble compounds from microalgae.

Limnospira indica PCC8005 growth in photobioreactor: model and simulation of the ISS and ground experiments.

The Arthrospira-B experiment is the first experiment in space ever allowing the online measurements of both oxygen production rate and growth rate of Limnospira indica PCC8005 in batch photobioreactors running on-board ISS. Four bioreactors were integrated in the ISS Biolab facility. Each reactor was composed of two chambers (gas and liquid) separated by a PTFE membrane and was run in batch conditions. Oxygen production was monitored by online measurement of the total pressure increase in the gas chamber. The experiments are composed of several successive batch cultures for each reactor, performed in parallel on ISS and on ground. In this work, a model for the growth of the cyanobacterium Limnospira indica PCC8005 (also known as Arthrospira or spirulina) in these space membrane photobioreactors was proposed and the simulation results obtained are compared to the experimental results gathered in space and on ground. The photobioreactor model was based on a light transfer limitation model, already used to describe and predict the growth and oxygen production in small to large scale ground photobioreactors. It was completed by a model for pH prediction in the liquid phase allowing assessment of the pH increase associated to the bicarbonate consumption for the biomass growth. A membrane gas-liquid transfer model is used to predict the gas pressure increase in the gas chamber. Substrate limitation is considered in the biological model. A quite satisfactory fit was achieved between experimental and simulation results when a suitable mixing of the liquid phase was maintained. The data showed that microgravity has no first order effect on the oxygen production rate of Limnospira indica PCC8005 in a photobioreactor operating in space in zero gravity conditions.

Optimal cultivation towards enhanced biomass and floridean starch production by Porphyridium marinum.

Optimal conditions for maximal biomass and starch production by the marine red microalgae Porphyridium marinum were investigated. Box-Behnken Design was used to model the effect of light intensity, NaNO3 concentration and salinity on the growth of microalgae but also on their starch and protein contents. These three factors increased biomass production by 13.6% in optimized conditions. A maximum starch production (140.21 µg·mL-1), 30.6% higher than that of the control, was attained at a light intensity of 100 µmol photons·m-2·s-1, a NaNO3 concentration of 1 g·L-1 and a NaCl concentration of 20 g·L-1. FT-IR spectroscopy was used to estimate the biochemical composition (carbohydrate accumulation) of P. marinum and revealed significant changes (P < 0.05) depending on culture conditions. FT-IR analysis highlighted also that the culture conditions leading to highest starch production by P. marinum corresponded to lowest sulfated polysaccharide and protein contents.

New horizons in culture and valorization of red microalgae.

Research on marine microalgae has been abundantly published and patented these last years leading to the production and/or the characterization of some biomolecules such as pigments, proteins, enzymes, biofuels, polyunsaturated fatty acids, enzymes and hydrocolloids. This literature focusing on metabolic pathways, structural characterization of biomolecules, taxonomy, optimization of culture conditions, biorefinery and downstream process is often optimistic considering the valorization of these biocompounds. However, the accumulation of knowledge associated with the development of processes and technologies for biomass production and its treatment has sometimes led to success in the commercial arena. In the history of the microalgae market, red marine microalgae are well positioned particularly for applications in the field of high value pigment and hydrocolloid productions. This review aims to establish the state of the art of the diversity of red marine microalgae, the advances in characterization of their metabolites and the developments of bioprocesses to produce this biomass.

Enhanced B-phycoerythrin production by the red microalga Porphyridium marinum: A powerful agent in industrial applications.

The production of B-phycoerythrin (B-PE) from the red microalga Porphyridium marinum was optimized before to purify it and subsequently study its antioxidant activities. NaNO3, K2HPO4 and metal traces concentrations of the culture medium, and luminosity parameters were chosen, according to the Plackett-Burman design, as the most influent factors on the B-PE production by P. marinum. The optimization of these factors according to the Box-Behnken plan gave a maximum of B-PE production equal to 40 mg/g dry weight under the following conditions: NaNO3 = 3.4 g/L; K2HPO4 = 0 g/L; light intensity = 70 µmol photons/m2/s and metal solution = 1.5 mL/L. The B-PE also showed an interesting capacity to chelate Fe3+ (IC50 = 13.91 ±â€¯0.21 µg/mL) and a significant reducing power (OD700nm = 0.485 ±â€¯0.011 at 100 µg/mL). The present study reports the antioxidant potential of purified B-PE from P. marinum that could be potentially used as a source of bioactive protein for a wide range of cosmetic and pharmaceutical applications.

Rheological and functional properties of asafoetida gum.

The asafoetida gum was extracted and purified from oleo-gum-resin of Ferula assa foetida root and characterized by high pressure anions exchange chromatography after acidic hydrolysis. It was composed of Gal:Ara:Rha:GlcA with the ratio 11.5:5.0:2.1:1.0. This monosaccharide composition was found similar to that of a commercial Arabic gum which exhibited a Gal:Ara:Rha:GlcA ratio of 11.7:5.4:3.2:1.0. As the Arabic gum is currently used for its emulsifying properties, the two gums were evaluated for their functional and rheological behaviors. Surface and interfacial tensions values were lower for asafoetida gum compared to Arabic gum. Critical micelle concentration was achieved at concentrations of 0.5% w/w and 1% w/w for asafoetida and Arabic gums, respectively. Values of emulsion capacity, emulsion stability and foaming properties were considerably higher for asafoetida gum in contrast to emulsion activity index that was lower than that of Arabic gum. As those of Arabic gum, solutions of asafoetida gum (2-30% w/w) exhibited Newtonian flow behavior at shear rates between 1 and 500 s-1. Apparent viscosities of Arabic and asafoetida gums were close and logically decreased by increasing temperature (10-80 °C). Higher viscosities were achieved at higher pH and CaCl2 concentrations.

Extraction and characterization of an alginate from the Iranian brown seaweed Nizimuddinia zanardini.

Sodium alginate from Nizimuddinia zanardini (an Iranian brown algae) was extracted with acid and alkaline solutions, partially and totally hydrolyzed and analyzed for its biochemical composition. 1H NMR spectroscopy, SEC-MALLS, HPAEC and FT-IR were performed to determine its structure and its physico-chemical properties. This alginate has a M/G ratio of 1.1, a molecular weight of 103 kDa, a polydispersity index of 1.22, and an intrinsic viscosity of 342 mL/g. Its antioxidant activity was tested by DPPH radical scavenging showing its potential for food preservation. Rheological properties of solutions of this alginate with concentrations between 1 and 5% (w/v) in water and 0.5 M NaCl were investigated indicating a Newtonian fluid type behaviour in water and a shear thinning fluid type behaviour in NaCl solutions.

Structural characterization and thermal behavior of a gum extracted from Ferula assa foetida L.

The gum asafoetida, an oleo-gum-resin from root of Ferula assa foetida, was extracted through alcoholic procedure followed by water extraction and then biochemically characterized using colorimetric assays, Fourier infrared spectroscopy, gas chromatography coupled to mass spectrometry, and 1D and 2D nuclear magnetic resonance. The gum was mainly composed of carbohydrates (67.39% w/w) with a monosaccharide distribution of 11.5: 5.9: 2.3: 1 between Gal, Ara, Rha and GlcA (molar ratio) and proteins (arabinogalactan protein). The polysaccharide consisted of a (1→3)-ß-d-galactan backbone ramified predominantly from O-6 but also from O-4 and O-4,6. Side chains included terminal-α-l-Araf, terminal-α-l-Rhap, (1→3)-α-l-Araf, (1→5)-α-l-Araf, terminal-ß-d-Galp, ß-d-GlcA and traces of (1→4)-ß-d-GlcA. X-ray diffraction pattern showed a semi crystalline microstructure. Thermal behavior of the gum was evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) revealed temperatures below and upper 200°C as dominant regions of weight loss.

Effects of nutritional conditions on growth and biochemical composition of Tetraselmis sp.

BACKGROUND: This study aimed to maximize biomass concentration, biomass productivity and biochemical composition of the marine microalga Tetraselmis sp. METHODS: In the current study, Box-Behnken Design was used to model the effect of NaNO3, NaH2PO4, metals and vitamins in the F/2 medium on the growth, total chlorophylls, carotenoids and starch contents. The total chlorophylls content was quantified by spectrophotometry. The FT-IR spectroscopy was used to estimate the biochemical compositions of Tetraselmis sp. grown under both optimized medium culture for starch production and standard culture medium. RESULTS: Finalized NaNO3 (1.76 mM), NaH2PO4 (0.018 mM), metals (1500 µL.L-1) and vitamins (312.5 µL.L-1) concentrations, generated an increase in biomass concentration up to 5.72 g.L-1 which contributed to an increase about 2.4-fold than that of the standard conditions of biomass productivity (408.57 mg.L-1.day-1). The maximum value of carotenoids content (0.3 mg.g DW-1) was achieved at the highest level of all factors. The total chlorophylls content reached also its maximum (5.18 mg.g DW-1) at high nitrate (1.76 mM), phosphate (0.054 mM), metals and vitamins concentrations, while the maximum starch content (42% DW) was achieved with low nitrate and phosphate concentrations (0.58 mM and 0.027 mM) and with metals and vitamins limitations. Thus, the nitrogen, phosphorus, metals and vitamins limitations led to divert the metabolism for the starch biosynthesis. CONCLUSIONS: The high biomass concentration productivity and starch production make Tetraselmis sp. strain a good candidate for biotechnological applications.

Production, extraction and characterization of microalgal and cyanobacterial exopolysaccharides.

The current interest of scientific but also industrial communities for the exploitation of microalgae is correlated with the development of specific culture and downstream processes. These processes are currently in progress, and the increasing of knowledges led to emergence of high value compounds such as pigments, proteins, polysaccharides, unsaturated fatty acids, enzymes and others. Exopolysaccharides from microalgae have been at this time poorly explored despite their original structural features associated with specific biological and physico-chemical properties. This situation could be changed in a next future with the lowering of costs for microalgae exploitation and the increasing of literature giving the structural data and specific properties of these biopolymers. The aim of this review is to do the state of the art of processes for exopolysaccharide production and extraction from microalgal biomass and the analytical strategies for their characterization.

Improvement of exopolysaccharide production by Porphyridium marinum.

With the aim to optimize the production of exopolysaccharide (EPS) by Porphyridium marinum, cultures in photobioreactors were conducted on a modified Provasoli medium (P) and compared to a new medium (Pm) with an elemental composition of N0.0205S0.0597P0.005. Cultivation on this medium allowed the increase of EPS concentration up to 2.5gL(-1), without modification of the EPS productivity (0.096gL(-1)) and EPS structure. In a second time, photosynthetic activity of the strain was monitored as a function of irradiance and temperature, allowing improvement of kinetic parameters of growth and EPS production. A semi-continuous culture, carried out with the Pm medium, an optimal irradiance and temperature of respectively 360µmolphotonsm(-2)s(-1) and 28°C led to an EPS process productivity of 0.031gh(-1) instead of 0.020gh(-1) in batch culture.

Haematococcus pluvialis soluble proteins: Extraction, characterization, concentration/fractionation and emulsifying properties.

A water-soluble matrix was extracted from green vegetative Haematococcus pluvialis through high-pressure cell disruption either at native pH (5.7) or with pH shifting to neutral (7). The resulting supernatant is mainly composed of carbohydrates and proteins, with the highest yield of proteins obtained at neutral pH (73±2% of total biomass proteins). The key emulsification properties of the proteins isolated in neutral supernatant (emulsification capacity (EC): 534±41mLoilg(-1) protein, emulsification stability (ES): 94±3% and emulsification activity index (EAI): 80±1m(2)g(-1)) were comparable to the native supernatant values (EC: 589±21mLoilg(-1) protein, ES: 84±3% and EAI: 75±1m(2)g(-1)). Confronted to sodium caseinate (EC: 664±30mLoilg(-1) protein, ES: 63±4%, and EAI: 56±4m(2)g(-1)) these results highlighted the strong potential of proteins isolated from H. pluvialis as emulsifier agent. Moreover, experiments have shown that the stability of emulsions obtained from supernatants is due to the proteins rather than the carbohydrates.

Structural Characterization and Biological Activities of Polysaccharides from Olive Mill Wastewater.

Olive mill wastewater (OMWW), the main waste product of olive oil extraction process, was investigated as a source of polysaccharides. The yield of alcohol insoluble residue (AIR) was 20.5 % based on the dry matter of OMWW. Extraction with water gave water soluble (WSF) and insoluble (WIF) fractions from AIR with yields of 13.3 % (w/w) and 3.7 % (w/w) based on the dry matter, respectively. Chemical composition and monosaccharide analysis indicated that glucose was the main monosaccharide of these extracts in addition to galactose, arabinose, rhamnose, and galacturonic acid. Prebiotic and antioxidant activities of polysaccharidic fractions from OMWW were evaluated. Results gave evidence for their scavenging capacity toward the 2,2'-diphenyl-1-picrylhydrazyle (DPPH) (IC50 value of 89.43 µg/mL) and hydroxyl radicals (IC50 value of 158.70 µg/mL), resistance toward artificial human gastric juice, and ability to be fermented by Lactobacilli strains.

Optimisation of culture parameters for exopolysaccharides production by the microalga Rhodella violacea.

A unicellular Rhodophyte was identified by sequencing of its 18S rRNA encoding gene as belonging to the Rhodella violacea specie. With the objective to optimise the production of biomass and exopolysaccharide by this strain, effects of irradiance, pH and temperature on its photosynthetic activity were investigated. In a second time a stoichiometric study of the well-known f/2 medium led to its supplementation in N and P to increase biomass and then exopolysaccharide yields when the strain was cultivated in photobioreactors. The use of optimal conditions of culture (irradiance of 420 µE/m(2)/s, pH of 8.3 and temperature of 24 °C) and f/2 supplemented medium led to significant increases of biomass and exopolysaccharide productions. The structural characterisation of the produced exopolysaccharide revealed that it was sulphated and mainly composed of xylose. The different culture conditions and culture media tested had no significant impact on the structure of produced exopolysaccharides.